WO 2006/021817 PCT/GB2005/050134 M&C Folio: WPP290168 Treatment of eye disorders 5 FIELD OF THE INVENTION The present invention relates to methods and compositions for the treatment of eye disorders; in particular but not exclusively to the treatment of glaucoma. In preferred embodiments, the invention relates to the use of RNAi technology to downregulate the expression of aqueous formation genes and aqueous outflow 10 genes. Methods and compositions for the treatment of eye disorders are also provided. BACKGROUND OF THE INVENTION RNAi as a tool to downregulate gene expression 15 Gene targeting by homologous recombination is commonly used to determine gene function in mammals, but this is a costly and time-consuming process. Alternatively, the functions of many genes can be determined after mRNA inhibition with ribozyme or antisense technologies. Although successful in some situations these technologies have been difficult to apply universally. The 20 advent of siRNA-directed "knockdown" has sparked a revolution in somatic cell genetics, allowing the inexpensive and rapid analysis of gene function in mammals. Establishing a convenient and reliable method to knock-out gene expression at 25 the mRNA level has been a recurrent theme in molecular biology over the last 15 years. In efforts to generate loss-of function cells or organisms, various molecules that included, for example, antisense sequences, ribozymes, and chimeric oligonucleotides have been tested, but the design of such molecules was based on trial and error, depending on the properties of the target gene.
WO 2006/021817 PCT/GB2005/050134 2 Moreover, the desired effects were difficult to predict, and often only weak suppression achieved (Braasch & Corey, 2002). After the discovery of the phenomenon in plants in the early 1990s, in 1998 5 Andy Fire and Craig Mello for the first time demonstrated with the worm Caenorhabditis elegans that dsRNA (double-stranded RNA) may specifically and selectively inhibit gene expression in an extremely efficient manner (Fire et al., 1998). In their experiment, the sequence of the first strand (the so-called sense RNA) coincides with that of the corresponding region of the target messenger 10 RNA (mRNA). The second strand (antisense RNA) is complementary to this mRNA. The resulting dsRNA turned out to be far more (several orders of ignitude) efficent than the~ corespondin -sinledstradd RNA moldcuess (ii particular, antisense RNA). Fire et al., 1998 named the phenomenon RNAi for RNA interference. This powerful gene silencing mechanism has been shown to 15 operate in several species among most phylogenetic phyla. RNAi begins when an enzyme named DICER encounters dsRNA and chops it into pieces called small-interfering RNAs or siRNAs. This protein belongs to the RNase III nuclease family. A complex of proteins gathers up these RNA remains 20 and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as target mRNA (for review see Bosher & Labouesse, 2000). The RNAi phenomenon (Akashi et al., 2001) might be summarized as follows: 25 a Step 1: dsRNA recognition and scanning process. e Step 2: dsRNA cleavage through RNase III activity and production of siRNAs. - Step 3: association of the siRNAs and associated factors in RISC complexes. * Step 4: recognition of the complementary target mRNA. - Step 5: cleavage of the target mRNA in the centre of the region 30 complementary to the siRNA. * Step 6: degradation of the target mRNA and recycling of the RISC complex.
WO 2006/021817 PCT/GB2005/050134 3 In trying to apply the RNAi phenomenon as a technology for gene knockdown, it was soon realized that mammalian cells have developed various protective phenomena against viral infections that could impede the use of this approach. 5 Indeed, the presence of extremely low levels of viral dsRNA triggers an interferon response, resulting in a global non-specific suppression of translation, which in turn triggers apoptosis (Williams, 1997, Gil & Esteban, 2000). In 2000, a first attempt with dsRNA resulted in the specific inhibition of 3 genes 10 (MmGFP under the control of the Elongation Factor la, E-cadherin, and c-mos) in the mouse oocyte and early embryo. Translational arrest, and thus a PKR ripohse, was Tidtob0s td aitheembryos continued-to-develop (Wianny& Zernicka-Goetz, 2000). One year later, research at Ribopharma AG (Kulmbach, Germany) first demonstrated the functionality of RNAi in mammalian cells. 15 Using short (20-24 base pairs) dsRNAs - which are called SIRPLEXm- they specifically switched off genes even in human cells without initiating the acute phase response. Similar experiments carried out later by other research groups (Elbashir et at, 2001; Caplen et al., 2001) further confirmed these results. 20 A year later, Paddison et al. (Paddison et al, 2002) tried to use small RNAs folded in hairpin structures to inhibit the function of specific genes. This work was inspired by previous studies showing that some genes in Caenorhabditis elegans naturally regulate other genes through RNAi by coding for hairpin structured RNAs. Tested in a variety of normal and cancer human and mouse 25 cell lines, short hairpin RNAs (shRNAs) are able to silence genes as efficiently as their siRNA counterparts. Moreover, shRNAs exhibit better reassoclation kinetics in vivo than equivalent duplexes. Even more important, these authors generated transgenic cell lines engineered to synthesize shRNAs that exhibit a long-lasting suppressing effect throughout cell divisions (Eurogentec). Recently, 30 another group of small RNAs (also comprised in the range of 21-25 nt) was shown to mediate downregulation of gene expression. These RNAs, known as WO 2006/021817 PCT/GB2005/050134 4 small temporally regulated RNAs (stRNAs), have been described in Caenorhabditis elegans were they regulate timing of gene expression during development. It should be noted that stRNAs and slRNAs, despite obvious similarities, proceed through different modes of action (for review see Banerjee 5 & Slack, 2002. In contrast with siRNAs, 22 nt long stRNAs downregulate expression of target mRNA after translational initiation without affecting mRNA integrity. Recent studies indicate that the two stRNAs first described in nematodes are the members of a huge family with hundreds of additional micro-RNAs (miRNAs) existing in metazoans (Grosshans & Slack, 2002). 10 Scientists have initially used RNAi in several systems, including Caenorhabditis e/egans, Drosophila thypnsomses" atnd various other inhrtbli rates: Moreoer using this approach, several groups have recently presented the specific suppression of protein biosynthesis in different mammalian cell lines 15 specifically in HeLa cells - showing that RNAi is a broadly applicable method for gene silencing in vitro. Based on these results, RNAi has rapidly become a well recognized tool for validating (identifying and assigning) gene functions. RNA interference employing short dsRNA oligonucleotides will, moreover, permit to decipher the function of genes being only partially sequenced. RNAi will 20 therefore become inevitable in studies such as: * Inhibition of gene expression at the post-transcriptional level in eukaryotic cells. In this context, RNAi is a straight-forward tool to rapidly assess gene function and reveal null phenotypes. - Development of the RNAi technology for use in post-implantation embryos. 25 - The predominant economic significance of RNA interference is established by its application as a therapeutic principle. As so, RNAi may yield RNA-based drugs to treat human diseases. Glaucoma 30 Glaucoma is one of the leading causes of blindness. Approximately 15% of cases of blindness world-wide result from glaucoma. The most common type, WO 2006/021817 PCT/GB2005/050134 5 primary open-angle glaucoma, has a prevalence of 1/200 in the general population over 40 years of age. Glaucoma has been simply defined as the process of ocular tissue destruction 5 caused by a sustained elevation of the Intra Ocular Pressure (IOP) above its normal physiological limits. It is becoming increasingly clear that many forms of glaucoma have a genetic component, and much current research is focused on identifying chromosomal 10 regions and genes that contribute to glaucoma. It is likely that the aetiology of OAG is multifactorial, resulting from a combination of mutations in more than one gen d~as yet unidentified enviionhental fatrs~ With regard to juvenile and adult-onset OAG, several loci have been identified. However, only one gene is known, namely the myocilin / TIGR (trabecular meshwork inducible 15 glucocorticoid response) gene at the GLC1A locus on chromosome 1q21-q31. More than thirty mutations of this gene have been identified in ethnically diverse populations worldwide. Studies have shown that it is responsible for only about 5% of OAG overall (See reviews in Wirtz & Samples, 2003, and Khaw et a], 2004a). 20 Pathogenesis Most glaucomas are characterised by an elevated IOP, although the level of elevation may vary. In those glaucomas in which the elevation is initially low (i.e., open angle glaucoma, melanocytic glaucoma) and some secondary 25 glaucoma, retinal ganglion cell and optic nerve damage are slow to progress. In angle-closure glaucoma the sudden high rise in IOP often renders the eye blind, undoubtedly primarily due to a cessation of axoplasmic flow at the level of the lamina cribrosa, 30 In human studies, it has been widely accepted that tissue ischaemia has a part to play in the initiation or progression of the optic disc damage that occurs in WO 2006/021817 PCT/GB2005/050134 6 glaucoma. Retinal ganglion cell degeneration may be necrosis, but the possibility that it is apoptosis triggered by the rise in IOP is plausible, and the respective roles of nitric oxide and glutamate are thought to be relevant during progression of the disease (For a recent review on the subject see Osborne et 5 al, 2003). Treatment Although several aetiologies are involved in the glaucoma complex, the absolute determinant in therapy selection is the amount of primary and/or induced 10 change in pressure within the iridocorneal angle. Curnif heapiesinclud- indications r .surteries aimdat ld e.1fig this pressure, although the pathophysiological mechanisms by which elevated IOP leads to neuronal damage in glaucoma are unknown. 15 Medical suppression of an elevated IOP can be attempted using four types of drugs: the aqueous formation suppressors (among them, carbonic anhydrase inhibitors, beta-adrenergic blocking agents, or alpha2-adrenoreceptor agonists) miotics (i.e. parasympathomimetics - cholinergics-, or anticholinesterase 20 inhibitors); uveoscleral outflow enhancers; and the hyperosmotic agents (that produce an osmotic pressure gradient across the blood/aqueous barrier within the ciliary epithelium). All four are used in the treatment of glaucoma, the first three commonly as emergency treatment and in long term control while the hyperosmotic agents are invaluable as emergency and preoperative treatment. 25 A fifth category of drugs, the neuroprotection agents, is beginning to emerge as an important possible addition to medical therapy. Indeed, observation that the NOS and glutamate levels. are elevated in glaucoma and that they are involved in retinal ganglion cell necrosis or apoptosis has raised the possibility of neuroprotective therapies and even neuroregeneration. Thus NOS inhibitors, 30 exciting amino acid antagonists, glutamate receptor antagonists, apoptosis inhibitors and calcium channel blockers are all potential candidates in the 7 development of future glaucoma therapies. The calcium channel blockers may reduce the effect of impaired microcirculation to the optic nerve head whilst potentially increasing outflow facility at the level of the trabecular cells. 5 Reviews of various eye disorders and their treatments are given in the references, in particular in Bunce (2005), Costagliola (1995, 2000), Cullinane (2002), Sakaguchi (2002), Shah (2000), and Wang (2005). Currently our existing therapies must fall short of the mark and the practical 10 difficulties associated with the assessment of outflow facility, the accurate monitoring of therapy and the complexity of surgical techniques all combine to confound the prognosis. The overriding factor in all glaucoma is the degeneration of the retinal ganglion cell, thus neuroprotection through effective ocular hypotension is the essential requirement of any therapy we utilise (for a 15 recent review on the subject, see Khaw et al 2004b). All references, including any patents or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states 20 what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in Australia or in any other 25 country. In the claims of this application and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or 30 "comprising" is used in an inclusive sense, i.e. to specify the presence of the 712123_1 (GHMatters) 7a stated features but not to preclude the presence or addition of further features in various embodiments of the invention. BRIEF SUMMARY OF THE INVENTION 5 In the present invention we describe a method for the treatment of eye conditions characterised by an altered IOP in animals, including humans. In particular, the eye conditions may include glaucoma, uveitis, and inflammation. The method is based on the downregulation of expression of genes involved in aqueous formation or aqueous outflow in the eye. Downregulation may be 10 effected by the use of double stranded nucleic acid moieties, named siNA or small interfering NA that are directed at interfering with the mRNA expression of various candidate genes. The siNA are preferably siRNA, although modified nucleic acids or similar chemically synthesised entities are also included within the scope of the invention. 15 Preferred embodiments of the invention relate to topical application of siNA. Embodiments of the invention also provide pharmaceutical compositions for use in the treatment of eye conditions. The invention may be used within the fields 712i23_1 (GHMattes) WO 2006/021817 PCT/GB2005/050134 8 of local eye treatments, of target genes involved in glaucoma pathogenesis, as well as the use of chemically synthesized entities to treat animal (including humans) diseases. 5 In addition to the treatment of glaucoma, the present method is also suitable for the treatment of other diseases of the anterior chamber of the eye. In particular, the method may be applied to the treatment of diseases characterised by altered aqueous formation or outflow in the eye. Examples of conditions which may be treated include local conditions such as infections or 10 inflammations, and general conditions such as uveitis or expression of systemic diseases. Further, certain embodiments of the invention provide treatment for .d tit tiathy ............ DETAILED DESCRIPTION OF THE INVENTION 15 Target genes In the present invention, we define a list of target genes, whose expression levels may alter IOP. These genes can fall within the groups of genes involved in aqueous formation or the group of genes involved in aqueous outflow. Here Is a list of our target genes: 20 . Carbonic Anhydrases II, IV and XII " Adrenergic Receptors: betal and 2 and alpha 1A, 1B and ID . Acetylcholinesterase . Cyclooxygenases 1 and 2 25 - ATPases: alphal, alpha2, alpha3, betal, beta2 . Endothelial Leukocyte Adhesion Molecule I (ELAM-1) . Angiotensin System: Angiotensin II, Angiotensin II Converting Enzymes (ACE I and ACE II), Angiotensin II Receptors (ATRI and ATR2) and Renin . Cochlin 30 WO 2006/021817 PCT/GB2005/050134 9 Design of siNA Although the mechanisms for RNAi remain unknown, the steps required to generate the specific dsRNA oligonucleotides are clear. It has been shown that dsRNA duplex strands that are 21-26 nucleotides in length work most 5 effectively in producing RNA interference. Selecting the right homologous region within the gene is also important. Factors such as the distance from start codon, the G/C content and the location of adenosine dimers are important when considering the generation of dsRNA for RNAi. One consequence of this, however, is that one may need to test several different sequences for the most 10 efficient RNAi and this may become costly. I. 1999, Tuschi et a. deciphafed the silencinig effete f siRNAs showing that their efficiency is a function of the length of the duplex, the length of the 3'-end overhangs, and the sequence in these overhangs. Based on this founder work, 15 Eurogentec recommends that the target mRNA region, and hence the sequence of the siRNA duplex, should be chosen using the following guidelines: Since RNAi relies on the establishment of complex protein interactions, it is obvious that the mRNA target should be devoided of unrelated bound factors. 20 In this context, both the 5' and 3' untranslated regions (UTRs) and regions close to the start codon should be avoided as they may be richer in regulatory protein binding sites. The sequence of the siRNA is therefore selected as follows: . In the mRNA sequence, a region located 50 to 100 nt downstream of the 25 AUG start codon or upstream of stop codon is selected. . In this region, the following sequences are searched for: AA(N19), CA(N19). . The G/C percentage for each identified sequence is calculated. Ideally, the G/C content is 50 % but it must less than 70 % and greater than 30 %. . Preferably, sequences containing following repetitions are avoided: AAA, 30 CCC, GGG, 1T, AAAA, CCCC, GGGG, TT1T.
WO 2006/021817 PCT/GB2005/050134 10 - An accessibility prediction according to the secondary structure of the mRNA is carried out as well. . A BLAST is also performed (i.e. NCBI ESTs database) with the nucleotide sequence fitting best the previous criteria to ensure that only one gene will 5 be inactivated. In order to maximize the result's interpretation, the following precautions should be taken when using siRNAs: * Always test the sense and antisense single strands in separate experiments. 10 e Try a scramble siRNA duplex. This should have the same nucleotide composition as your siRNA but lack significant sequence homology to any other gene.(in.udi.-..... ........................ . .-.............. - If possible, knock-down the same gene with two independent siRNA duplexes to control the specificity of the silencing process. 15 Practically, each of the selected genes is introduced as a nucleotide sequence in a prediction program that takes into account all the variables described above for the design of optimal oligonucleotides. This program scans any mRNA nucleotide sequence for regions susceptible to be targeted by siRNAs. The 20 output of this analysis is a score of possible siRNA oligonucleotides. The highest scores are used to design double stranded RNA oligonucleotides (typically 21 bp long, although other lengths are also possible) that are typically made by chemical synthesis. 25 In addition to siRNA, modified nucleotides may also be used. We plan to test several chemical modifications that are well known in the art. These modifications are aimed at increasing stability or availability of the siNA. Examples of suitable modifications are described in the publications referenced below, each of which is incorporated herein by reference. 30 WO 2006/021817 PCT/GB2005/050134 11 Studies show that replacing the 3'-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two -nucleotide 3'-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with 5 deoxyribonucleotides has been reported to be well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir 2001). In addition, Elbashir et al. also report that substitution of siRNA with 2' 0- methyl nucleotides completely abolishes RNAi activity. 10 Affinity modified nucleosides as described in W02005/044976 may be used. This publication describes oligonucleotides comprising nucleosides modified so as to havs sincreasedrdscrsa§adaffiiityfortheirfomipleientary nucleotide in..... the target mRNA and/or in the complementary siNA strand. 15 GB2406568 describes alternative modified oligonucleotides chemically modified to provide improved resistance to degradation or improved uptake. Examples of such modifications include phosphorothioate internucleotide linkages, 2'-0 methyl ribonucleotides, 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, and 20 inverted deoxyabasic residue incorporation. W02004/029212 describes oligonucleotides modified to enhance the stability of the siRNA or to increase targeting efficiency. Modifications include chemical cross linking between the two complementary strands of an siRNA and chemical 25 modification of a 3' terminus of a strand of an siRNA. Preferred modifications are internal modifications, for example, sugar modifications, nucleobase modifications and/or backbone modifications. 2'-fluoro modified ribonucleotides and 2'-deoxy ribonucleotides are described. 30 W02005/040537 further recites modified oligonucleotides which may be used in the Invention.
WO 2006/021817 PCT/GB2005/050134 12 As well as making use of dsNA and modified dsNA, the present invention may use short hairpin NA (shNA); the two strands of the siNA molecule may be connected by a linker region, which may be a nucleotide linker or a non 5 nucleotide linker. In addition to siNA which is perfectly complementary to the target region, degenerate siNA sequences may be used to target homologous regions. W02005/045037 describes the design of siNA molecules to target such 10 homologous sequences, for example by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences. n instances where mismatches are identified, non-caronical base pairs (for example, mismatches and/or wobble bases) can be used to generate siNA molecules that target more than one gene sequence. In a non 15 limiting example, non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting sequences for differing targets that share sequence homology. As such, one advantage of using siNAs of the invention is that a single siNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is 20 conserved between homologous genes. In this approach, a single siNA can be used to inhibit expression of more than one gene instead of using more than one siNA molecule to target different genes. Preferred siNA molecules of the invention are double stranded. A siNA molecule 25 of the invention may comprise blunt ends, that is, ends that do not include any overhanging nucleotides. In one embodiment, an siNA molecule of the invention can comprise one or more blunt ends. In preferred embodiments, the siNA molecules have 3' overhangs. siNA molecules of the invention may comprise duplex nucleic acid molecules with 3' overhangs of n nucleotides 30 (5 nt1). Elbashir (2001) shows that 21-nucleotide siRNA duplexes are most active when containing 3'-terminal dinucleotide overhangs.
WO 2006/021817 PCT/GB2005/050134 13 Candidate oligonucleotides are further filtered for interspecies sequence conservation in order to facilitate the transition from animal to human clinical studies. In preferred embodiments of the invention, conserved oligonucleotides 5 are used; this allows a single oligonucleotide sequence to be used in both animal models and human clinical trials. GenBank Accession Numbers corresponding to our selected human target genes are shown in Figure 1. In some of these genes, alternative splicing produces a 10 family of transcripts that differ in exon content. The present invention allows individual targeting of each transcript form. Selected oligonucleotide sequences against which RNAI is directed are shown in Figure 2. Displayed sequences are the DNA sequences targeted by the siNA. 15 Therefore, the invention would make use of NA duplexes with sequences complementary to the indicated DNA sequences. The sequences shown in Figure 2 are not limiting. As a matter of fact, target DNA need not necessarily be preceded by AA or CA. Further, target DNA could 20 be constituted by sequences included in Figure 2 flanked by any contiguous sequence. In vitro and animal studies. Obtaining siRNA duplexes 25 RNAs are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Substitution of one or both strands of a siRNA duplex by 2'-deoxy or 2'-0 methyl oligoribonucleotides abolished silencing in fly extract (Elbashir et al. 2001). In mammalian cells, however, it seems possible to substitute the sense 30 siRNA by a 2'-O-methyl oligoribonucleotide (Ge et al 2003).
WO 2006/021817 PCT/GB2005/050134 14 Most conveniently, siRNAs are obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality and costs. In general, 21-nt RNAs are not too difficult to synthesize and are readily provided in a quality suitable for RNAi. 5 Suppliers of RNA synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK), Qiagen (Germany), Ambion (USA) and Invitrogen (Scotland). The previous custom RNA 10 synthesis companies are entitled to provide siRNAs with a license for target validation. In particular, our siRNA suppliers are Ambion, Dharmacon and Invitrogni companies that offerthetraditiariaI custar chemical"~synthesis service for siRNA, and supply the siRNA with HPLC purification and delivered in dry form along with RNase-free water. A central web-based resource for RNAi 15 and siRNA methodologies, along with links to additional siRNA related products and services, can be found on the website of above-mentioned suppliers. An annealing step is necessary when working with single-stranded RNA molecules. It is critical that all handling steps be conducted under sterile, Rnase 20 free conditions. To anneal the RNAs, the oligos must first be quantified by UV absorption at 260 nanometres (nm). The following protocol based on Elbashir et al. (2001) is then used for annealing: " Separately aliquot and dilute each RNA oligo to a concentration of 50 pM. " Combine 30 pl of each RNA oligo solution and 15 pl of 5X annealing buffer. 25 Final buffer concentration is: 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate. Final volume is 75 pl. * Incubate the solution for 1 minute at 90 *C, centrifuge the tube for 15 seconds, let sit for 1 hour at 37 *C, then use at ambient temperature. The solution can be stored frozen at -20 *C and freeze-thawed up to 5 times. The 30 final concentration of siRNA duplex is usually 20 pM. Alternatively, already annealed dsRNAs may be purchased from the suppliers.
WO 2006/021817 PCT/GB2005/050134 15 Chemically modified nucleic acids may also be used. For example, an overview of the types of modification which may be used is given in WO03/070744, the contents of which are incorporated herein by reference. Particular attention is 5 drawn to pages 11 to 21 of this publication. Other possible modifications are as described above. The skilled person will be aware of other types of chemical modification which may be incorporated into RNA molecules. In vitro system 10 To check the specificity of the siRNA interference different cell cultures that express the target genes were employed. The cells used for these experiments were: rabbit~ ~~5 -piyrintad tiliary epitheliuMi cells NPE, hum-an~ ciliary epithelium cells OMDC, and human embryonic kidney cells HEK293. The cells are incubated with the corresponding siRNA duplexes, and analysis of the 15 downregulation of expression of the target gene is carried out. For linking siRNA knockdown to specific phenotypes in cultured cells, it is necessary to demonstrate the reduction of targeted protein or at least demonstrate the reduction of the targeted mRNA. 20 mRNA levels of the target gene can be quantitated by real-time quantitative PCR (RT-PCR). Further, the protein levels can be determined in a variety of ways well known in the art, such as Western blot analysis with specific antibodies to the different target allow direct monitoring of the reduction of targeted protein. 25 Transfection of siRNA duplexes Several examples of techniques well known in the art are as follows: We can perform a single transfection of siRNA duplex using a cationic lipid, such as RNAiFect Transfection Reagent (Qiagen) and Lipofectamine 2000 Reagent 30 (Invitrogen) and assay for silencing 24, 48 and 72 hours after transfection.
WO 2006/021817 PCT/GB2005/050134 16 A typical transfection protocol can be performed as follows: For one well of a 6 well plate, we transfect using 100nM as final concentration of siRNA. Following RNAiFect protocol, we seed, the day before transfection, 2-4 x 105 cells per well In 3ml of an appropriate growth medium, containing DMEM, 10% serum, 5 antibiotics and glutamine, and incubate cells under normal growth conditions (37 0 C and 5% C0 2 ). On the day of transfection, cells have to be at 30-50% confluence. We dilute 15ul of 20uM siRNA duplex (corresponding to 100 nM final concentration) in 85ul of Buffer EC-R, to give a final volume of 100ul, and mix by vortexing. For complex formation, we add 19 ul of RNAiFect Transfection 10 Reagent to the diluted siRNA and mix by pipetting or vortexing. After incubating the samples for 10-15 minutes at room temperature to allow formation of transfection comp lexes, we add the coimplee's-dpro-wise~ontothec'elis with~2.9 ml of fresh growth medium low in antibiotics. After swirling the plates to ensure uniform distribution of the transfection complexes, we incubate the cells under 15 their normal growth conditions. The day after, the complexes are removed and fresh and complete growth medium is added. To monitor gene silencing, cells are collected at 24, 48 and 72 hours post-transfection. The Lipofectamine 2000 Reagent protocol is quite similar. The day before transfection, we seed 2-4 x 105 cells per well in 3ml of an appropriate growth medium, containing DMEM, 20 10% serum, antibiotics and glutamine, and incubate cells under normal growth conditions (37 0 C and 5% C0 2 ). On the day of transfection, cells have to be at 30-50% confluence. We dilute 12.5ul of 2OuM siRNA duplex (corresponding to 100 nM final concentration) in 250ul of DMEM, to give a final volume of 262.5ul, and mix. Also, 6ul of Lipofectamine 2000 is diluted in 250ul of DMEM and 25 mixed. After a 5 minutes incubation at room temperature, the diluted oligomer and the diluted Lipofectamine are combined to allow complex formation during a 20 minutes incubation at room temperature. Afterwards, we add the complexes drop-wise onto the cells with 2 ml of fresh growth medium low in antibiotics and mix gently by rocking the plate back and forth, to ensure 30 uniform distribution of the transfection complexes. We incubate the cells under their normal growth conditions and the day after, the complexes are removed WO 2006/021817 PCT/GB2005/050134 17 and fresh and complete growth medium is added. To monitor gene silencing, cells are collected at 24, 48 and 72 hours post-transfection. The efficiency of transfection may depend on the cell type, but also on the 5 passage number and the confluency of the cells. The time and the manner of formation of siRNA-liposome complexes (e.g. inversion versus vortexing) are also critical. Low transfection efficiencies are the most frequent cause of unsuccessful silencing. Good transfection is a non-trivial issue and needs to be carefully examined for each new cell line to be used. Transfection efficiency 10 may be tested transfecting reporter genes, for example a CMV-driven EGFP expression plasmid (e.g. from Clontech) or a B-Gal expression plasmid, and then assessed by pfiase contras and/or fluorescence microsco py thehext day. Testing of siRNA duplexes 15 Depending on the abundance and the life time (or turnover) of the targeted protein, a knock-down phenotype may become apparent after 1 to 3 days, or even later. In cases where no phenotype is observed, depletion of the protein may be observed by immunofluorescence or Western blotting. 20 After transfections, total RNA fractions extracted from cells were pre-treated with DNase I and used for reverse transcribed using a random primer. PCR is amplified with a specific primer pair covering at least one exon-exon junction in order to control for amplification of pre-mRNAs. RT/PCR of a non-targeted mRNA is also needed as control. Effective depletion of the mRNA yet 25 undetectable reduction of target protein may indicate that a large reservoir of stable protein may exist in the cell. Alternatively, RealTime PCR amplification can be used to test in a more precise way the mRNA decrease or disappearance. Real-time reverse-transcriptase (RT) PCR quantitates the initial amount of the template most specifically, sensitively and reproducibly. Real 30 time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle. This signal increases in direct WO 2006/021817 PCT/GB2005/050134 18 proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. 5 To verify the interference pattern of the differentially expressing genes identified in the cell cultures, qRT-PCR was performed according to the manufacturer protocol.. For quantitative RT-PCR (qRT-PCR), approximately 250 ng of total RNA were used for reverse transcription followed by PCR 10 amplification with specific primers for each gene in reaction mixture containing Master SYBR Green I. Basic PCR conditions comprised an initial step of 30 minutes at 91 foWed~ by40 cyclesdf~5Tat 95 0 C, 10 s at 62 0 C and 15~s at 720C. Specific primer sequences corresponding to each target gene were used. Quantification of b-actin mRNA was used as a control for data normalization. 15 Relative gene expression comparisons work best when the gene expression of the chosen endogenous/internal control is more abundant and remains constant, in proportion to total RNA, among the samples. By using an invariant endogenous control as an active reference, quantitation of an mRNA target can be normalised for differences in the amount of total RNA added to each 20 reaction. Pharmaceutical formulations The present invention may comprise the administration of one or more species of siNA molecule simultaneously. These species may be selected to target one 25 or more target genes. The siNA molecules of the invention and formulations or compositions thereof may be administered directly or topically (e. g., locally) to the eye as is generally known in the art. For example, a siNA molecule can comprise a 30 delivery vehicle, including liposomes, for administration to a subject. Carriers and diluents and their salts can be present in pharmaceutically acceptable WO 2006/021817 PCT/GB2005/050134 19 formulations. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins poly (lactic 5 co-glycolic) acid (PLGA) and PLCA microspheres, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors. In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed. with polyethyleneimine and derivatives thereof, such as polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or 10 polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine
(PEI-PEG
triGAL) derivatives. A siNA molecule of the invention may be complexed with membrane disruptive agents and/or a cationic lipid or helper lipid molecule. 15 Delivery systems which may be used with the invention include, for example, aqueous and non aqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and non aqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as 20 solubilizers, permeation enhancers (e. g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e. g. , polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. 25 A pharmaceutical formulation of the invention is in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Other factors are known in the art, and include considerations such as toxicity and forms that 30 prevent the composition or formulation from exerting its effect.
WO 2006/021817 PCT/GB2005/050134 20 The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical 5 art. For example, preservatives, stabilizers, dyes and flavouring agents can be provided. These include sodium benzoate, sorbic acid and esters of p hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used. 10 A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms)of a disease state The phiatseuticaly effectivedosedepends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal 15 under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1mg/kg and 100 mg/kg body weight/day of active ingredients is administered. 20 The formulations of the invention can be administered in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. Formulations can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily 25 suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavouring agents, colouring agents or preservative 30 agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic WO 2006/021817 PCT/GB2005/050134 21 pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, 5 sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such 10 coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example,a tfime delay~ materiat suh as ~lycefyl monostearate or glycery distearate can be employed. 15 Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. 20 Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl- methylcellulose, sodium alginate, 25 polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or 30 condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or WO 2006/021817 PCT/GB2005/050134 22 condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, 5 one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin. Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a 10 mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavong agents can be~~added tprovide sMPtabie oral prepratis" These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid. 15 Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are 20 exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, can also be present. Pharmaceutical compositions of the invention can also be in the form of oil-in water emulsions. The oily phase can be a vegetable oil or a mineral oil or 25 mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for 30 example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavouring agents.
WO 2006/021817 PCT/GB2005/050134 23 Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavouring and colouring agents. 5 The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been 10 mentioned above. Sterile injecle preparation can a1s6 be a sterile injectablie soltio r suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents 15 that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. 20 The nucleic acid molecules of the invention can also be administered in the form of suppositories, e. g. , for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that Is solid at ordinary temperatures but liquid at the rectal 25 temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols. Nucleic acid molecules of the invention can be administered parenterally In a sterile medium. The drug, depending on the vehicle and concentration used, 30 can either be suspended or dissolved in the vehicle. Advantageously, adjuvants WO 2006/021817 PCT/GB2005/050134 24 such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle. It is understood that the specific dose level for any particular subject depends 5 upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy. 10 For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the ania tekes in therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to 15 the feed or drinking water. The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication 20 can increase the beneficial effects while reducing the presence of side effects. Alternatively, certain siNA molecules of the invention can be expressed within cells from eukaryotic promoters. Recombinant vectors capable of expressing the siNA molecules can be delivered and persist in target cells. Alternatively, vectors 25 can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells 30 ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.
WO 2006/021817 PCT/GB2005/050134 25 Animal studies The New Zealand rabbit is the gold standard in experimental platforms designed to study IOP. It is easy to handle and it has a big eye, similar in size 5 to the human organ. In addition, present equipment to measure IOP is not suited to use in animals with small eyes such as mice or rats. Finally, rabbits have an IOP (around 23 mm Hg) that can be brought down to up to 40% its value using local commercial hypotensive medication. Thus, although it is possible to generate rabbit glaucoma models (for example, surgically blocking 10 episclerotic veins or artificially occluding the trabecular meshwork), we have used normotensive rabbits since, in our hands, the pharmacological decrease in ILOP can be easily and reiproducibly mesired Experimental protocol 15 Normotensive New Zealand White rabbits (males, 2-3 kg) were used. The animals were kept in individual cages with free access to food and water. They were submitted to artificial 12 hours light/darkness cycles, to avoid uncontrolled circadian oscillations of IOP Animal handling and treatment were carried out in accordance with the European Communities Council Directive (86/609/EEC) and 20 the statement of the Association for Research in Vision and Ophthalmology on the Use of Animals in Ophthalmic and Vision Research. The drugs were typically administered by instilling a small volume (typically 40 pL) on the corneal surface. Contralateral eyes were treated with the vehicle 25 alone and could be used as controls in each experiment lest there is a sympathy phenomenon with the other eye. Multiple experiments in the same animal should be abolished. IOP measurements were done using a contact tonometer (TONOPEN XL, 30 Mentor, Norwell, Massachusetts). The TonoPen tonometer is very convenient due to its reliability and small size. Measurements with this instrument were WO 2006/021817 PCT/GB2005/050134 26 performed delicately applying the tonometer's sensor to the corneal surface. This device has been shown to be the tonometer of choice for measuring intraocular pressures within the range of 3 to 30 mm Hg in rabbits (Abrams et al., 1996). All measurements fell within this interval: the mean baseline value of 5 intraocular pressure was 17.0 ± 0.39 mm Hg (n = 100). Because IOP changes from the night to day, all the experiments were performed at the same time to allow IOP more stable and permit an objective comparison with vehicle treatment. In order to avoid distress to the animal, rabbits were topically anesthetized (oxibuprocaine/tetracaine, 0.4%/1%, in a saline solution (1/4 v:v). 10 The solution was applied (10 pl) to the cornea before each measurement of intraocular pressure was made. siRNA or saline was applied topically to the cornea in volumes of 40 pl. The standard protocol for the siRNA application in rabbit was as follows. Doses 15 of siRNA in saline solution (0.9% w/v) to a final volume of 40ul, were applied to one eye each day during four consecutive days. The opposite eye was taken as a control and 40pl of sterile saline (0.9% w/v) were instilled on it, at the same time points. The IOP was measured before each application and at 2h, 4h and 6h following the instillation, during 10 days. Maximum responses were observed 20 between second and third day. To compare the effect of siRNA with other hypotensive compounds, Xalatan (latanoprost 0.005%) and Trustop (Dorzolamide 2%) were assayed and IOP measured in the same conditions. Results 25 Example 1 In vitro assays. To determine the inhibition of the different targets involved in glaucoma using RNAi technology, the first step was to perform experiments in cell cultures. For each target, several siRNAs were designed using a specific software according 30 to the rules described before. Those with the best characteristics were selected to be tested. The siRNAs were applied to cell cultures, such as NPE, OMDC and WO 2006/021817 PCT/GB2005/050134 27 HEK293. The effect of siRNAs over the target gene was analyzed by real time PCR and semi-quantitative PCR according to standard protocols. The gene target transcript levels were normalized using actin as housekeeping gene. Table I below shows representative results of real time PCR experiments for 5 some of the target genes described previously. The values represent the mean of the percentage of siRNA interference over each gene expression once normalized with the control cells and their standard deviations. Compared to the control cells, the level of the different transcripts at both 24 and 48h time points was significantly reduced after the siRNA treatment. In the Table are 10 included some of the different siRNAs that were tested and their different efficacies in the interference of the target gene. The siRNAs used in the Table correspond to the listed human siRNA targets givei Fingue 2~ a f6olows. AC2: siRNA1: rabbit sequence homologous to human SEQ. ID. 73 siRNA2: rabbit sequence identical to human SEQ. ID. 54 15 siRNA3: rabbit sequence identical to human SEQ, ID. 66 PTGS1 siRNA1: rabbit sequence homologous to human SEQ. ID. 353 siRNA2: rabbit sequence homologous to human SEQ. ID. 369 PTGS2 siRNA1: rabbit sequence identical to human SEQ. ID. 426 siRNA2: rabbit sequence homologous to human SEQ. ID. 421 20 siRNA3: rabbit sequence homologous to human SEQ. ID. 477 WO 2006/021817 PCT/GB2005/050134 28 % of gene transcript level 24h 48h Target AC2 siRNA1 - 76.25±12.60 84.57±14.70 siRNA2 37.97±9.78 61.45±9.62 siRNA3 35.30±9.73 i 51.14±16.49 .. PTGSI RIRNA 4225±13r6e- 42.68±17.00 siRNA2 1 34.98±14.33 26.30±10.91 PGS sFIRNAI 86±2487.71. -siRNA2 I 81.00±13.54 I 66.85±18.67 Ta l .......... ..... . .... ,_I.-.-..... ... - _'".--- ........ 4....... ... . 83.... ±16-- -- -. 6...... ..... ........ ...... T b I-:- siINAtreatrnent -reduces -the--levels. of -target-gene-transcripts. RNA-was prepared-from ...... cells treated with different siRNAs for 24 and 48h. The samples were analyzed by real time PCR 5 using specific primers. The values show the mean expression levels of different transcripts normalized to actin relative to cell control. Figure 3 shows some representative semi-quantitative gels for some of the targets described above. The decrease of the gene expression for each target 10 gene depends on the efficiency in siRNA silencing. For each target, the most effective siRNA obtained by in vitro studies was administered to, the animal model. RNA was prepared from cells treated with different siRNAs. The samples were analyzed by semi-quantitative PCR using specific primers. The figure shows a representative semi-quantitative gel for Beta Adrenergic Receptor 2 15 expression (A) and other for Acetylcholinesterase expression (B). M: MW Marker; C: Control cells; TC: Transfection Control; 1: siRNA1; 2: siRNA2; 3: sIRNA3; NC: Negative Control. The expression levels for each target depends on the efficiency in siRNA silencing. The siRNAs used in the Figure correspond to the human targets given in Figure 2 as follows: 20 Panel A (Beta Adrenergic Receptor 2) 1: rabbit sequence homologous to human SEQ. ID. 122 2: rabbit sequence identical to human SEQ. ID. 125 3: rabbit sequence homologous to human SEQ. ID. 139 Panel B (Acetylcholinesterase) WO 2006/021817 PCT/GB2005/050134 29 1: rabbit sequence homologous to human SEQ. ID. 162 2: rabbit sequence homologous to human SEQ. ID. 177 Example 2 In vivo assays. 5 Previously to the siRNA therapeutical application, the in vivo assays were validated to determine the proper siRNA delivery. Those siRNAs selected by the in vitro assays were applied to the animal model, following the protocol previously described. To avoid the effect of IOP 10 fluctuations due to circadian cycles, all the applications were performed at the same time. To determine the siRNA effect, intraocular pressures (IOPs) were measured as previously mentioned Since glaucoma pathology presents an increase in the intraocular pressure, the 15 aim was to obtain a decrease in its levels following siRNA application. Most of the results for the different targets showed a significant decrease in IOP levels comparing with controls and also with commercial drugs (Latanoprost and Dorzolamide) and the animals treated with vehicle alone 20 (negative control) didn't present any significant change in their IOP baseline. The data are summarized in table II where values represent the mean of the maximum percentage of IOP reduction after siRNA treatment once normalized and their standard deviations. The decrease in IOP was statistically significant for all the treated targets. These results indicate that siRNAs and commercial 25 drugs act in a similar way, reducing IOP levels around 20%, although siRNAs present a more maintained effect. No secondary effects were observed in the animals along the experimental protocols. The siRNAs used in these experiments correspond to the human targets given in Figure 2 as follows: AC2: rabbit sequence homologous to human SEQ. ID. 73 30 AC4: rabbit sequence identical to human SEQ. ID. 5 AC12: rabbit sequence identical to human SEQ. ID. 522 WO 2006/021817 PCT/GB2005/050134 30 ADRBI: rabbit sequence identical to human SEQ. ID. 105 ADRB2: rabbit sequence homologous to human SEQ. ID. 139 ADRA1A: rabbit sequence homologous to human SEQ. ID. 546 ADRA1B: rabbit sequence homologous to human SEQ. ID. 619 5 ACHE: rabbit sequence homologous to human SEQ. ID. 189 PTGS1: rabbit sequence homologous to human SEQ. ID. 322 PTGS2: rabbit sequence identical to human SEQ. ID. 426 SELE: rabbit sequence homologous to human SEQ. ID. 262 ACE1: rabbit sequence homologous to human SEQ. ID. 866 10 AGTR1: rabbit sequence homologous to human SEQ. ID. 705 AGTR2: rabbit sequence identical to human SEQ. ID. 774 ATP1A1: rabbit sequence identical to hiian~SEQ.ID.1399 ATP1B2: rabbit sequence identical to human SEQ. ID. 1820 AC2 24.84±3.41 AC4 14.47±5.00 AC12 24.30±1.29 ADRB1 _ 28.04±2.98. ... ADRB2 21.18 1.88 ADRAlA 19.51±1.04 ADRAIB 17.48±1.30 ACHE 125.25±2.70 PTGS1 14.62±1.93.... PTGS2 23.78±2.27. SELE :21.80±1.74 IACE1 17.51±1.28 ........... AGTRI 9.72±1.35 AGTR2 11.22i1.53 ATP1A1 118.13±1.39 ATPB2 116.32±0.91 Latanopgs 25.4±5.24 Dorzolamide 16.41±2.38 15 Table II: Effect of sIRNAs on the reduction of iOP in normotensive New Zealand rabbit. The values represent the mean of the percentage of IOP reduction over the control (contralateral eye with vehicle alone) and their standard error (SEM).
WO 2006/021817 PCT/GB2005/050134 31 REFERENCES Abrams LS, Vitale S, Jampel HD. Comparison of three tonometers for measuring intraocular pressure in rabbits. Invest Ophthalmol Vis Sci. 1996 Apr;37(5):940 5 4. Akashi H, Miyagishi M, Taira K. Suppression of gene expression by RNA interference in cultured plant cells. Antisense Nucleic Acid Drug Dev, 2001, 11(6):359-67. Banerjee D, Slack F. Control of developmental timing by small temporal RNAs: a 10 paradigm for RNA-mediated regulation of gene expression. Bioessays, 2002, 24(2):119-29. hatfacharya SK Annangudi SP,SalonidiRG, Kuchtey"RW; Pathey NS,Crabb JW. Cochlin deposits in the trabecular meshwork of the glaucomatous DBA/23 mouse. Exp Eye Res. 2005a May;80(5):741-4. 15 Bhattacharya SK, Rockwood E, Smith SD, Bonilha VL, Crabb JS, Kuchtey RW, Robertson NG, Peachey NS, Morton CC, Crabb JW. Proteomics reveal Cochlin deposits associated with glaucomatous trabecular meshwork. J Biol Chem. 2005b Feb 18;280(7):6080-4. Epub 2004 Dec 3. Bosher JM, Labouesse M. RNA interference: genetic wand and genetic 20 watchdog. Nat Cell Biol, 2000, 2(2):E31-6. Braasch DA, Corey DR. Novel antisense and peptide nucleic acid strategies for controlling gene expression. Biochemistry, 2002, 41(14):4503-10 Bunce C, Hitchings RA, Van Duijn CM, De Jong PT, Vingerling JR. Associations between the deletion polymorphism of the angiotensin 1-converting enzyme 25 gene and ocular signs of primary open-angle glaucoma. Graefes Arch Clin Exp Ophthalmol. 2005 Apr;243(4):294-9. Epub 2004 Oct 13. Caplen, N.J., Parrish, S., Imani, F., Fire, A. & Morgan, R.A. Specific inhibition of gene expression by small double stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA, 2001, 98: 9742-9747. 30 Costagliola C, Verolino M, De Rosa ML, Iaccarino G, Ciancaglini M, Mastropasqua L. Effect of oral losartan potassium administration on intraocular WO 2006/021817 PCT/GB2005/050134 32 pressure in normotensive and glaucomatous human subjects. Exp Eye Res. 2000 Aug;71(2):167-71. Costagliola C, Di Benedetto R, De Caprio L, Verde R, Mastropasqua L. Effect of oral captopril (SQ 14225) on intraocular pressure in man. Eur J Ophthalmol. 5 1995 Jan-Mar;5(1):19-25. Cullinane AB, Leung PS, Ortego J, Coca-Prados M, Harvey BJ. Renin-angiotensin system expression and secretory function in cultured human ciliary body non pigmented epithelium. Br J Ophthalmol. 2002 Jun;86(6):676-83. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 10 22-nucleotide RNAs. Genes Dev, 2001, 15(2):188-200. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetiterference by dolesstranded~RNA inc Cieio.habditis~ legans. Nature, 1998, 391(6669):806-11. Ge Q, McManus MT, Nguyen T, Shen CH, Sharp PA, Eisen HN, Chen J. RNA 15 interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc NatI Acad Sci U S A., 2003; 100(5):2718-23. Gil J, Esteban M. Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis, 2000, 5(2):107-14. 20 Grosshans H, Slack FJ. Micro-RNAs: small is plentiful. J Cell Biol, 2002, 156(1):17-21. Hara H, Ichikawa M, Oku H, Shimazawa M, Araie M. Bunazosin, a selective alpha1-adrenoceptor antagonist, as an anti-glaucoma drug: effects on ocular circulation and retinal neuronal damage. Cardiovasc Drug Rev. 2005 25 Spring;23(1):43-56. Khaw PT, Shah P, Elkington AR. Glaucoma-1: diagnosis. BMJ, 2004a, 328:97-9. Khaw PT, Shah P, Elkington AR. Glaucoma-2: treatment. BMJ, 2004b, 328:156 8. Osborne NN, Chidlow G, Wood J, Casson R. Some current ideas on the 30 pathogenesis and the role of neuroprotection in glaucomatous optic neuropathy. Eur J Ophthalmol. 2003 Apr; 13 Suppl 3:519-26.
WO 2006/021817 PCT/GB2005/050134 33 Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev, 2002, 16(8):948-58. Sakaguchi H, Takal S, Sakaguchi M, Sugiyama T, Ishihara T, Yao Y, Miyazaki M, 5 Ikeda T. Chymase and angiotensin converting enzyme activities in a hamster model of glaucoma filtering surgery. Curr Eye Res. 2002 May;24(5):325-31. Shah GB, Sharma S, Mehta AA, Goyal RK. Oculohypotensive effect of angiotensin-converting enzyme inhibitors in acute and chronic models of glaucoma. 3 Cardiovasc Pharmacol. 2000 Aug;36(2):169-75. 10 Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev., 1999; 13(24):3191 Wang RF, Podos SM, Mittag TW, Yokoyoma T. Effect of CS-088, an angiotensin ATI receptor antagonist, on intraocular pressure in glaucomatous monkey eyes. 15 Exp Eye Res. 2005 May;80(5):629-32. Epub 2005 Jan 4. Wianny F, Zernicka-Goetz M. Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol, 2000, 2(2):70 5. Williams BR. Role of the double-stranded RNA-activated protein kinase (PKR) in 20 cell regulation. Biochem Soc Trans, 1997, 25(2):509-13. Wirtz MK, Samples JR. The genetic loci of open-angle glaucoma. Ophthalmol Clin North Am. 2003 16:505-14